We propose to study the mechanisms of oxygenase catalyzed O2 activation and insertion reactions. We have purified, characterized, and, in many cases, crystallized a group of Fe-containing aromatic dioxygenases which appear to be good systems with which to investigate all recognized types of oxygen activation chemistry. Aromatic dioxygenase catalyze cleavage of O2 and insertion of both oxygen atoms into the aromatic being of their organic substrates, resulting in either ring opening or loss of aromaticity. The substrates of these enzymes serve as focal points in pathway of bacterial degradation of aromatic compounds, thus they are of substantial environmental significance. Similar enzymes catalyze essential steps in mammalian biosynthetic pathways. The enzymes proposed for study include: protocatechuate 2,3-dioxygenase, protocatechuate 3.5- dioxygenase (e,4-PCD), protocatechuate 4,5-dioxygenase (4,5-PCD), homoprotocatechuate 2,3-dioxygenase, catechol 2,3-dioxygenase, gentisate 1,2-dioxygenase (1,2-GTD), and benzoate 1,2-dioxygenase, (1,2-BZD). Past studies have lead to the development of models of the mechanisms of their position of ring cleavage relative to the two vicinal OH groups of the substrate. It is proposed that intradiol dioxygenase (e.g. 3,4-PCD) activate substrate for attack by O2, while estradiol dioxygenase (e.g. 4,5-PCD) activate O2 for attack on the substrate. 1,2-GTD appears to utilize a mechanism analogous to that of the extradiol dioxygenase. We now plan to use this wide range of enzymes to test and refine the mechanistic hypotheses. We also plan to develop a mechanistic model for the role of the mononuclear iron center in 1,2-BZD, an enzyme that adds both oxygens from O2 to benzoate without cleaving the ring. The work will involve studies in 4 areas: structure, kinetics, spectroscopy, and site directed mutagenesis. The refined X-ray crystal structure of 3,4- PCD and 3 recently solved structures of substrate analog complexes will serve as the basis for the structural and the site directed mutagenesis studies. Similar crystallographic and molecular genetic studies have been initiated for the other dioxygenases. Stopped flow, stopped freeze, low temperature, and photolysis techniques will be used to study the binding of substrates and the formation of oxy-intermediates. The structure of the Fe environment of these intermediates will be investigated using optical, EPR, integer spin EPR, resonance Raman, NMR, EXAFS, and Mossbauer spectroscopies. Analogs for the substrates and O2 will be labeled with O, C, and N to study their interactions with the Fe through EPR-detected hyperfine interaction. These studies will be complemented by coordinated and/or collaborative studies of toluene dioxygenase, and enzyme mechanistically similar to 1,2-BZD, and isopenicillin N-synthase, which our spectroscopic studies indicate has an iron center similar to those of the estradiol dioxygenase. This work should yield fundamental information about the chemistry of oxygenases, O2, and iron.